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Abstract:

The invention provides a quick method for detecting the genus of Bacillus
in samples from oil reservoirs, which comprise: collecting samples from
oil reservoirs; inactivating microbes except for spore-forming Bacillus
in the samples through high temperature; collecting spores from the
samples; incubating the collected spores in a medium, and stimulating to
resurrect Bacillus under in situ temperatures of the oil reservoirs;
etecting the resurrected Bacillus in the cultures using molecular
biological techniques. The present invention provides an effective method
to detect the genus of Bacillus in samples from oil reservoirs, which is
conducive to the discovery of the functional Bacillus for microbial
enhanced oil recovery (MEOR) and to revealing the ecosystem of Bacillus
in oil reservoirs.

Claims:

1. A quick method for detecting the genus Bacillus in samples from oil
reservoirs, which comprises: (a) collecting samples from oil reservoirs;
(b) inactivating microbes except for spore-forming Bacillus in the
samples through high temperature; (c) collecting spores from the samples;
(d) incubating the collected spores in an appropriate medium, and
stimulating to resurrect the Bacillus under the in situ temperatures of
the oil reservoirs; (e) detecting the resurrected Bacillus in the
cultures using molecular biological techniques.

2. A quick method for detecting the genus Bacillus in samples from oil
reservoirs according to claim 1, wherein said reservoirs are various oil
reservoirs with different geological conditions and exploitation methods.

3. A quick method for detecting the genus Bacillus in samples from oil
reservoirs according to claim 2, wherein said samples are selected from
oil-water samples collected directly from the wellheads of injection
wells, or production wells, or formation water samples, or flowing-back
sample of the oil wells, or core samples from oil reservoirs.

4. A quick method for detecting the genus Bacillus in samples from oil
reservoirs according to claim 3, wherein said step (a) of collecting
samples comprising: oil-water samples are collected and put into sterile
plastic bottles, full-filled, sealed and record the in situ temperature
of the oil reservoirs of the collected samples.

5. A quick method for detecting the genus Bacillus in samples from oil
reservoirs according to claim 4, wherein said step (b) of inactivating
microbes except for spore-forming Bacillus in the samples through high
temperature comprising: a 500 ml of the oil-water sample is transferred
into a sterile flask in a sterile condition, and sealed with a sterile
ventilate membrane; and put the flask on an electric heater and keep the
oil-water sample boiling for 10 minutes, cool down at room temperature.

6. A quick method for detecting the genus Bacillus in samples from oil
reservoirs according to claim 5, wherein said step (c) of collecting
spores from the inactivating samples comprising: after cool down, the
inactivating oil-water sample is transferred into 50 ml sterile
centrifuge tubes in sterile condition and centrifuge at 10,000.times.g
for 15 minutes, remove the supernatant to a sterile container for medium
preparation; resuspend the pellets of precipitation in each centrifuge
tube with 1 ml sterile water, and combine the suspension from all the
centrifuge tubes into a 10 ml mixture; the mixture contains spores.

7. A quick method for detecting the genus Bacillus in samples from oil
reservoirs according to claim 6, wherein said step (d) of incubating the
collected spores in a medium, and stimulating to resurrect the Bacillus
under the in situ temperatures of the oil reservoirs comprising: the 10
ml mixture collected in step (c) is incubated into a 100 ml flask with 30
ml sterilized medium, seal the cover of the flask, cultivate to resurrect
the Bacillus at in situ temperature of the oil reservoir 10.degree. C. to
110.degree. C., from which the sample was collected, for 7-15 days at 150
rmp to obtain the resurrection medium; wherein said medium is compatible
with the environments from which samples were collected, and can provide
nutrients for stimulating spores, the medium (100 ml) includes: 2 g
molasses, 0.16 g (NH4)2HPO4, 0.2 g KNO3, constant the
volume with the supernatant collected in step 3 to 100 ml, and add 2 g
crude oil; autoclave at 121.degree. C. for 20 minutes, the crude oil is
from the oil reservoirs where the sample was collected.

8. A quick method for detecting the genus Bacillus in samples from oil
reservoirs according to claim 7, wherein said step (e) of detecting the
resurrected Bacillus in the cultures using molecular biological
techniques comprising: a 5 ml of the stimulating medium in the step (d)
is transferred into a sterile centrifuge tube in a sterile condition,
centrifuged at 10,000.times.g for 10 minutes for cell collection, remove
the supernatant and add 1 ml distilled water to resuspend the cells
sedimentation; DNA in the resuspension is extracted following the
manufacturer's protocol of the FastDNA Spin Kit for Soil, and the
extracted DNA is amplified by PCR using specific 16S RNA gene primers for
bacteria to construct clone library, the representative clones are
sequenced and are analysed to determine their phylogenetic affiliations.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a quick method for detecting
Bacillus in samples from oil reservoirs. More specifically, the present
invention relates to a method to reveal the diversity of Bacillus in oil
reservoirs, by the combination of culture-dependent microbiological
techniques and culture-independent molecular biological techniques.

BACKGROUND ART

[0002] With the detection and isolation of microbes in samples from oil
reservoirs, researchers found out that not all microbes in oil reservoirs
are harmful to oil exploitation, the metabolism products and activity of
some microbes in oil reservoirs can be use to enhance oil recovery.
Therefore, microbial enhanced oil recovery (MEOR) was proposed and
implemented as one of tertiary measures to enhance oil recovery.

[0003] The principal part of MEOR is potential microbes. According to
studies of functional microbes for MEOR, strains of Bacillus are
documented as a functional group of microbes for MEOR. Bacillus is able
to produce biosurfactant that can lower interfacial tension between crude
oil and aqueous liquid and rocks, so as to improve the mobility of the
crude oil and significantly increase the production of the oil; Bacillus
can increase the production of the oil by degrading long chain
hydrocarbons to decrease viscosity of crude oil and to improve the
mobility of crude oil; Bacillus can also increase the production of the
oil by producing polymer to selectively plug in oil reservoirs, so as to
promote the volumetric sweep efficiency of injected water.

[0004] For studies and field trials of MEOR, investigating microbes
inhabiting in oil reservoirs is a key and primary step to discover
potential microbes and design strategies of field trials.

[0005] Traditional culture-dependent microbial methods are applied by
researchers to isolate microbes from oil reservoirs and identify their
potential functions to MEOR. However, the procedures to enrich and
isolate microbes are complex, in most of cases, special media, special
carbon resources and tens of incubations cycle during procedure of
enrichment were needed. In addition, only a small percentage (less than
1%) (Amann et al., 1995) of viable microbes can be cultured on known
nutrient media using the culture-dependent traditional techniques.
Bacteria detected by traditional microbial methods are a minor part of
the microbes inhabiting in oil reservoirs.

[0006] Recently, molecular microbial ecology represents a development in
research methods, which contains utilizing techniques of molecular
biology to investigate the ecology of microbes and offers new techniques
to facilitate the detection of microbes. Researchers have investigated
microbial diversity in samples collected from various oil reservoirs
using culture-independent molecular biological methods.

[0007] Multiple groups of microorganisms with diverse phylogenetic
affiliations have routinely been detected from oil reservoirs. However,
an obscuring result from the studies based on culture-independent
molecular techniques was that, in most of clone libraries, few to no
clones or sequences detected were affiliated with the genus of Bacillus.
These results insert an impression to us that Bacillus is rare or absent
in these oil reservoirs.

[0008] When investigating microbial diversity in the original samples from
oil reservoirs based on culture-independent molecular biological methods,
DNA extraction from the spore-forming bacteria is more difficult than
from other microbes, in addition, molecular biological methods is unable
to detect a small proportion of 0.1-1% of bacteria (Head et al., 1998),
both of which might be the major reasons for neglection of the group of
Bacillus in clone libraries of samples from oil reservoirs.

[0009] The Bacillus is a group of Gram-positive, spore-forming bacteria,
which is a potential microbial group for MEOR. How to detect and enrich
the genus of Bacillus in samples from oil reservoirs is an urgent
question faced by us. Considering the disadvantages and advantages of
culture-dependent and culture-independent microbial methods and molecular
biological methods, we provide a method to quickly detect the genus
inhabiting in samples collected from oil reservoirs. This method is a
combination of the advantages of both culture-dependent and
culture-independent microbial methods and molecular biological methods.

CONTENTS OF THE INVENTION

[0010] It is an object of present invention to provide a method for
detecting the genus of Bacillus in samples from oil reservoirs based on
the advantages of both culture-dependent and culture-independent
microbial methods and molecular biological methods, which provides an
effective method to discover the functional Bacillus for microbial
enhanced oil recovery (MEOR) and reveal the ecosystem of Bacillus in oil
reservoirs.

[0011] According to one aspect, the present invention relates to a method
for enriching and detecting Bacillus in samples from oil reservoirs based
on culture-dependent coupled with culture-independent molecular
biological techniques, which comprise:

(1) collecting samples from oil reservoirs; (2) inactivating microbes
except for spore-forming Bacillus in the samples through high
temperature; (3) collecting spores from the samples; (4) incubating the
collected spores in an appropriate medium, and stimulating to resurrect
the Bacillus under the in situ temperatures of the oil reservoirs; (5)
detecting the resurrected Bacillus in the cultures using molecular
biological techniques.

[0012] Wherein, said reservoirs are various oil reservoirs with different
geological conditions and exploitation methods.

[0013] Wherein said samples include oil-water samples collected directly
from the wellheads of injection wells, production wells, formation water
samples, flowing-back sample of the oil wells, core samples from oil
reservoirs.

wherein, the step of collection samples comprising: an oil-water sample
are collected and put into a sterile plastic bottle, full filled and
sealed and recorded the in situ temperature of the oil reservoir.
wherein, the step of inactivating microbes except for spore-forming
Bacillus in the samples through high temperature is as follow: a 500 ml
of the oil-water sample is transferred into a 2 L sterile flask in a
sterile condition, and sealed with a sterile ventilate membrane. Put the
flask on an electric heater and keep the oil-water sample inside boiling
for 10 minutes, cool down at room temperature.

[0014] The step of collecting spores from the samples treated in step 2 is
that after cool down, the oil-water sample is transferred into 50 ml
sterile centrifuge tubes in sterile condition and centrifuged at
10,000×g for 15 minutes, remove the supernatant to a sterile flask
for medium preparation;

[0015] Then, resuspend the pellets of precipitation in each centrifuge
tube with 1 ml sterile water, and combine the suspension from the
centrifuge tubes into a 10 ml mixture; the mixture contains spores.

[0016] The next step consists in the incubation and cultivation for
resurrection. The 10 ml mixture collected in the first step is incubated
into a 100 ml flask with 30 ml sterilized appropriate medium, seal the
cover of the flask, cultivate to resurrect the Bacillus at in situ
temperature of the oil reservoir from which the sample was collected for
7-15 days to obtain the resurrection medium.

[0017] In the step of cultivation, the optimal temperature is the in situ
temperature of the oil reservoir from which the sample was collected.
Different oil reservoirs have different in situ temperatures, and the
range of the in situ temperature of the oil reservoirs is 10°
C.-110° C.

[0018] In the step of incubation and cultivation for resurrection, an
appropriate medium is needed. As used herein, an "appropriate medium" is
intended to mean a medium compatible with the environment from with the
sample was collected and can provide nutrients for spores stimulation. In
the invention for oil-water samples from oil reservoirs, the supernatant
collected in the step (3) can be the appropriate water containing
suitable ingredients for medium preparation. The medium (100 ml) includes
the supernatant collected in the step (3), 2 g molasses, 0.16 g
(NH4)2HPO4, 0.2 g KNO3 and 2 g crude oil, autoclave at 121° C. for
20 minutes. The crude oil is oil from the oil reservoirs where the sample
was collected.

[0019] The step of detecting microbes in the culture can based on
molecular biological techniques is as follow: a 5 ml of the culture is
transferred into a sterile centrifuge tube in sterile condition, and
centrifuge at 10,000×g for 10 minutes for cell collection, remove
the supernatant and add 1 ml distilled water to resuspend the cells
sedimentation. DNA in the resuspension were extracted following the
manufacturer's protocol of the FastDNA Spin Kit for Soil, and extracted
DNA were amplified by PCR using specific 16S RNA gene primers for
bacteria to construct clone library, the representative clones are
sequenced and are analysed to determine their phylogenetic affiliations.

[0020] Most strains of Bacillus in samples from oil reservoirs might have
been overlooked as their character of spore-forming and the disadvantages
of detecting methods. The present invention relates to a quick method to
detect and enrich Bacillus from samples collected from oil reservoirs
based on culture-dependent methods coupled with culture-independent
methods, which provide an effective method to discover the functional
Bacillus and to reveal the ecosystem of Bacillus in oil reservoirs.

DESCRIPTION OF FIGURES

[0021] FIG. 1 is a photo of a collected spore by the method of the
invention pictured by transmission electron microscope.

MODE OF CARRYING OUT THE INVENTION

[0022] Different oil reservoirs have different in situ temperatures. Eight
samples from four water-flooded oil reservoirs with different in situ
temperatures (two samples respectively are from an injection well and a
production well of each water-flooded oil reservoir), and two samples
from a polymer-flooded oil reservoir were used in the invention.

[0023] The investigations of microbial diversity in the five oil
reservoirs reported that few to no sequence of Bacillus were detected.
Results obtained in these examples determine effectiveness, extensity and
feasibility of the invention.

Example 1

Detection of Bacillus in the Samples from the Oil Reservoir with Low In
Situ Temperature

[0024] Two samples were collected from the No. 6 oil reservoir with an in
situ temperature of 25° C. in Xinjiang oil field (China). One
sample was collected from the injection well of T6186 and the other was
collected from the production well of T6073. The primary investigation of
microbial diversity was reported that few to no sequences were affiliated
with Bacillus in the both clone libraries constructed from the two
original samples.

[0025] According to the methods of the invention, the steps of detecting
the genus of Bacillus are as follows:

[0026] 1. Collecting samples from
the oil reservoir of No. 6. Oil-water samples were collected directly
from the wellheads of the injection well of T6186 and the production well
of T6073, and put into two sterile plastic bottles, full filled and
sealed, and recorded the in situ temperature of the oil reservoir of
25° C. after which, the experiments were carried out ASAP.

[0027]
2. Inactivating microbes except for spore-forming Bacillus in the samples
through high temperature. A 500 ml of each the oil-water sample was
transferred into a 2 L sterile flask in a sterile condition, and sealed
with a sterile ventilate membrane. Put the flask on an electric heater
with asestos net and keep the oil-water sample inside boiling for 10
minutes, cool down at room temperature.

[0028] 3. Collecting spores from
the samples. After cool down, the oil-water sample treated in step 2 was
transferred into 50 ml sterile centrifuge tubes in a sterile condition,
and centrifuged at 10,000×g for 15 minutes, remove the supernatant
to a sterile flask for medium preparation; resuspend the pellets of
precipitation in each centrifuge tube with 1 ml sterile water, and
combine the suspension from the centrifuge tubes into a 10 ml mixture;
the mixture contains spores.

[0029] FIG. 1 is a photo of a collected spore by the method of the
invention pictured by transmission electron microscope.

[0030] 4.
Incubating the collected spores in an appropriate medium, and stimulating
to resurrect the Bacillus under the in situ temperatures of the oil
reservoirs: The 10 ml mixture collected in step 3 was incubated into a
100 ml flask with 30 ml sterilized appropriate medium, sealed the cover
of the flask, cultivated to resurrect the Bacillus at the in situ
temperature of 25° C. of the oil reservoir for 7 days at 150 rmp
to obtain the resurrection medium.

[0031] Said medium is a medium compatible with the environment from with
the sample was collected and can provide nutrients for spores
stimulation. The medium (100 ml) includes the supernatant collected in
step 3, 2 g molasses, 0.16 g (NH4)2HPO4, 0.2 g KNO3 and 2 g crude oil,
autoclave at 121° C. for 20 minutes. The crude oil is oil from the
oil reservoir of No. 6.

[0032] 5. Detecting the Bacillus in the cultures
using molecular biological techniques. A 5 ml of the culture obtained in
step 4 was transferred into a sterile centrifuge tube in a sterile
condition, and centrifuged at 10,000×g for 10 minutes for cell
collection, removed the supernatant and added 1 ml distilled water to
resuspend the cells sedimentation. DNA in the resuspension were extracted
following the manufacturer's protocol of the FastDNA Spin Kit for Soil,
the extracted DNA were amplified by PCR using specific 16S RNA gene
primers for bacteria to construct clone library, the representative
clones were sequenced and were analysed to determine their phylogenetic
affiliations.

[0033] Two 16S rRNA gene clone libraries (N6PB and N6IB) were constructed
from the two cultures cultivated following the invention. 5 and 6 OTUs
were identified in clone libraries of N6PB and N6IB, respectively. 98.8%
of the sequences in clone library of N6PB were identified as Bacillus,
dominated by Bacillus sp. (Table 1). In clone library of N6IB, 95.4% of
sequences were also affiliated with Bacillus, dominated by Bacillus
flexus (Table 2).

Detection of the Bacillus in the Samples from the Oil Reservoir with
Mesothermal In Situ Temperature

[0034] Two samples were collected from the Yan 9 oil reservoir with a
mesothermal in situ temperature of 40° C. in Changing oil field
(China). One sample was collected from the injection well of L28-45 and
the other was collected from the production well of L28-46. The primary
investigation of microbial diversity in the two samples was reported that
few to no sequences were affiliated with Bacillus in the both clone
libraries constructed from the two original samples.

[0035] According to the methods of the invention, the steps of detecting
the genus of Bacillus are as follows:

[0036] 1. Collecting samples from
the oil reservoir of Yan 9. Oil-water samples were collected directly
from the injection well of L28-45 and the production well of L28-46, and
put into two sterile plastic bottles, full filled and sealed, in
addition, recorded the in situ temperature of the oil reservoir of
40° C., after which, the experiments were carried out ASAP.

[0037]
2. Inactivating microbes except for spore-forming Bacillus in the samples
through high temperature. A 500 ml of each the oil-water sample was
transferred into a 2 L sterile flask in a sterile condition, and sealed
with a sterile ventilate membrane. Put the flask on an electric heater
with asestos net and keep the oil-water sample inside boiling for 10
minutes, cool down at room temperature.

[0038] 3. Collecting spores from
the samples. After cool down, the oil-water sample treated in step 2 was
transferred into 50 ml sterile centrifuge tubes in a sterile condition,
and centrifuged at 10,000×g for 15 minutes, remove the supernatant
to a sterile flask for medium preparation; resuspend the pellets of
precipitation in each centrifuge tube with 1 ml sterile water, and
combine the suspension from the centrifuge tubes into a 10 ml mixture;
the mixture contains spores.

[0039] 4. Incubating the collected spores in
an appropriate medium, and stimulating to resurrect the Bacillus under
the in situ temperatures of the oil reservoirs. The 10 ml mixture
collected in step 3 was incubated into a 100 ml flask with 30 ml
sterilized appropriate medium, sealed the cover of the flask, cultivated
to resurrect the Bacillus at the in situ temperature of 40° C. of
the oil reservoir for 9 days at 150 rmp to obtain the resurrection
medium.

[0040] Said medium is a medium compatible with the environment from with
the sample was collected and can provide nutrients for spores
stimulation. The medium (100 ml) includes the supernatant collected in
step 3, 2 g molasses, 0.16 g (NH4)2HPO4, 0.2 g KNO3 and 2 g crude oil,
autoclave at 121° C. for 20 minutes. The crude oil is oil from the
oil reservoir of Yan 9.

[0041] 5. Detecting the Bacillus in the cultures
using molecular biological techniques. A 5 ml of the culture obtained in
step 4 was transferred into a sterile centrifuge tube in a sterile
condition, and centrifuged at 10,000×g for 10 minutes for cell
collection, removed the supernatant and added 1 ml distilled water to
resuspend the cells sedimentation. DNA in the resuspension were extracted
following the manufacturer's protocol of the FastDNA Spin Kit for Soil,
and the extracted DNA were amplified by PCR using specific 16S RNA gene
primers for bacteria to construct clone library, the representative
clones were sequenced and were analysed to determine their phylogenetic
affiliations.

[0042] Two 16S rRNA gene clone libraries (Y9PB and Y9IB) were constructed
from the two samples. 4 and 4 OTUs were identified in in Y9PB and Y9IB,
respectively. 100% of the sequences in clone library of Y9PB were
identified as Bacillus, dominated by Brevibacillus agri. (Table 3). In
clone library of Y9IB, 100% of sequences were also affiliated with
Bacillus, dominated by Paenibacillus sp. (Table 4).

Detection of the Bacillus in the Samples from the Oil Reservoir with High
In Situ Temperature

[0043] Two samples were collected from the J68 oil reservoir with a high
in situ temperature of 60° C. in Liaohe oil field (China). One
sample was collected from the injection well of J68-50 and the other was
collected from the production well of J68-51C. The primary investigation
of microbial diversity in the two samples was reported that few to no
sequences were affiliated with Bacillus in the both clone libraries
constructed from the two original samples.

[0044] 1. Collecting samples
from the oil reservoir of J68. Oil-water samples were collected directly
from the wellheads of the injection well of J68-50 and the production
well of J68-51C, and put into two sterile plastic bottles, full filled
and sealed, in addition, recorded the in situ temperature of the oil
reservoir of 60° C., after which, the experiments were carried out
ASAP.

[0045] 2. Inactivating microbes except for spore-forming Bacillus
in the samples through high temperature. A 500 ml of each the oil-water
sample was transferred into a 2 L sterile flask in a sterile condition,
and sealed with a sterile ventilate membrane. Put the flask on an
electric heater with asestos net and keep the oil-water sample inside
boiling for 10 minutes, cool down at room temperature.

[0046] 3.
Collecting spores from the samples. After cool down, the oil-water sample
treated in step 2 was transferred into 50 ml sterile centrifuge tubes and
centrifuged at 10,000×g for 15 minutes, remove the supernatant to a
sterile flask for medium preparation; resuspend the pellets of
precipitation in each centrifuge tube with 1 ml sterile water, and
combine the suspension from the centrifuge tubes into a 10 ml mixture;
the mixture contains spores.

[0047] 4. Incubating the collected spores in
an appropriate medium, and stimulating to resurrect the Bacillus under
the in situ temperatures of the oil reservoirs. The 10 ml mixture
collected in step 3 was incubated into a 100 ml flask with 30 ml
sterilized appropriate medium, sealed the cover of the flask, cultivated
to resurrect the Bacillus at the in situ temperature of 60° C. of
the oil reservoir for 10 days at 150 rmp to obtain the resurrection
medium.

[0048] Said medium is a medium compatible with the environment from with
the sample was collected and can provide nutrients for spores
stimulation. The medium (100 ml) includes the supernatant collected in
step 3, 2 g molasses, 0.16 g (NH4)2HPO4, 0.2 g KNO3 and 2 g crude oil,
autoclave at 121° C. for 20 minutes. The crude oil is oil from the
oil reservoir of J68.

[0049] 5. Detecting the Bacillus in the cultures
using molecular biological techniques. A 5 ml of the culture obtained in
step 4 was transferred into a sterile centrifuge tube in a sterile
condition, and centrifuged at 10,000×g for 10 minutes for cell
collection, removed the supernatant and added 1 ml distilled water to
resuspend the cells sedimentation. DNA in the resuspension were extracted
following the manufacturer's protocol for the FastDNA Spin Kit for Soil,
and the extracted DNA were amplified by PCR using specific 16S RNA gene
primers for bacteria to construct clone library, the representative
clones were sequenced and were analysed to determine their phylogenetic
affiliations.

[0050] Two 16S rRNA gene clone libraries (J68PB and J68IB) were
constructed from the two samples following the invention. 7 and 5 OTUs
were identified in clone libraries of J68PB and J68IB, respectively.
95.6% of the sequences in clone library of J68PB were identified as
Bacillus, dominated by Geobacillus thermodenitrificans (Table 5). In
clone library of J68IB, 100% of sequences were also affiliated with
Bacillus, dominated by Bacillus thermoamylovorans and Bacillus cereus
(Table 6).

Detection of the Bacillus in the Samples from the Oil Reservoir with Super
High In Situ Temperature

[0051] Two samples were collected from the V 4 oil reservoir with a super
high in situ temperature of 70° C. in Henan oil field (China). One
sample was collected from the injection well of V187 and the other was
collected from the production well of V 149. The primary investigation of
microbial diversity in the two samples was reported that few to no
sequences were affiliated with Bacillus in the both clone libraries
constructed from the two original samples.

[0052] 1. Collecting samples
from the oil reservoir of V 4. Oil-water samples were collected directly
from the injection well of V187 and the production well of V149, and put
into two sterile plastic bottles, full filled and sealed, in addition,
recorded the in situ temperature of the oil reservoir of 70° C.,
after which, the experiments were carried out ASAP.

[0053] 2.
Inactivating microbes except for spore-forming Bacillus in the samples
through high temperature. A 500 ml of each the oil-water sample was
transferred into a 2 L sterile flask in a sterile condition, and sealed
with a sterile ventilate membrane. Put the flask on an electric heater
with asestos net and keep the oil-water sample inside boiling for 10
minutes, cool down at room temperature.

[0054] 3. Collecting spores from
the samples. After cool down, the oil-water sample treated in step 2 was
transferred into 50 ml sterile centrifuge tubes in a sterile condition,
and centrifuged at 10,000×g for 15 minutes, remove the supernatant
to a sterile flask for medium preparation; resuspend the pellets of
precipitation in each centrifuge tube with 1 ml sterile water, and
combine the suspension from the centrifuge tubes into a 10 ml mixture;
the mixture contains spores.

[0055] 4. Incubating the collected spores in
an appropriate medium, and stimulating to resurrect the Bacillus under
the in situ temperatures of the oil reservoirs. The 10 ml mixture
collected in step 3 was incubated into a 100 ml flask with 30 ml
sterilized appropriate medium, sealed the cover of the flask, cultivated
to resurrect the Bacillus at the in situ temperature of 70° C. of
the oil reservoir for 12 days at 150 rmp to obtain the resurrection
medium.

[0056] Said medium is a medium compatible with the environment from with
the sample was collected and can provide nutrients for spores
stimulation. The medium (100 ml) includes the supernatant collected in
step 3, 2 g molasses, 0.16 g (NH4)2HPO4, 0.2 g KNO3 and 2 g crude oil,
autoclave at 121° C. for 20 minutes. The crude oil is oil from the
oil reservoir of V4.

[0057] 5. Detecting the Bacillus in the cultures
using molecular biological techniques. A 5 ml of the culture obtained in
step 4 was transferred into a sterile centrifuge tube in a sterile
condition, and centrifuged at 10,000×g for 10 minutes for cell
collection, removed the supernatant and added 1 ml distilled water to
resuspend the cells sedimentation. DNA in the resuspension were extracted
following the manufacturer's protocol of the FastDNA Spin Kit for Soil,
and the extracted DNA were amplified by PCR using specific 16S RNA gene
primers for bacteria to construct clone library, the representative
clones were sequenced and were analysed to determine their phylogenetic
affiliations.

[0058] Two 16S rRNA gene clone libraries (V4PB and V4IB) were constructed
from the two cultures cultivated following the invention. 5 and 5 OTUs
were identified in V4PB and V4IB, respectively. 100% of the sequences in
clone library of V4PB were identified as Bacillus, dominated by Bacillus
sp., and included a small percentage of Aneurinibacillus aneurinilyticus
(Table 7); In clone library of V4IB, 100% of sequences were also
affiliated with Bacillus, dominated by Bacillus pseudofirmus, and
Aneurinibacillus migulanus occupied a percentage of 14.6% (Table 8).

Detection of the Bacillus in the Samples from the Polymer-Flooded Oil
Reservoir

[0059] Two samples were collected from the N 2 oil reservoir, which is a
polymer-flooded oil reservoir with an in situ temperature of 40°
C. One sample was collected from the injection well of N2-1 and the other
was collected from the production well of N2-2. The primary investigation
of microbial diversity in the two samples was reported that no sequences
were affiliated with Bacillus in the both clone libraries constructed
from the two original samples.

[0060] 1. Collecting samples from the oil
reservoir of N 2. Oil-water samples were collected directly from the
injection well of N2-1 and the production well of N2-2, and put into two
sterile plastic bottles, full filled and sealed, in addition, recorded
the in situ temperature of the oil reservoir, after which, the
experiments were carried out ASAP.

[0061] 2. Inactivating microbes except
for spore-forming Bacillus in the samples through high temperature. A 500
ml of each the oil-water sample was transferred into a 2 L sterile flask
in a sterile condition, and sealed with a sterile ventilate membrane. Put
the flask on an electric heater with asestos net and keep the oil-water
sample inside boiling for 10 minutes, cool down at room temperature.

[0062] 3. Collecting spores from the samples. After cool down, the
oil-water sample treated in step 2 was transferred into 50 ml sterile
centrifuge tubes in a sterile condition and centrifuged at 10,000×g
for 15 minutes, remove the supernatant to a sterile flask for medium
preparation; resuspend the pellets of precipitation in each centrifuge
tube with 1 ml sterile water, and combine the suspension from the
centrifuge tubes into a 10 ml mixture; the mixture contains spores.

[0063] 4. Incubating the collected spores in an appropriate medium, and
stimulating to resurrect the Bacillus under the in situ temperatures of
the oil reservoirs. The 10 ml mixture collected in step 3 was incubated
into a 100 ml flask with 30 ml sterilized appropriate medium, sealed the
cover of the flask, cultivated to resurrect the Bacillus at the in situ
temperature of the oil reservoir for 15 days at 150 rmp to obtain the
resurrection medium.

[0064] Said medium is a medium compatible with the environment from with
the sample was collected and can provide nutrients for spores
stimulation. The medium (100 ml) includes the supernatant collected in
step 3, 2 g molasses, 0.16 g (NH4)2HPO4, 0.2 g KNO3 and 2 g crude oil,
autoclave at 121° C. for 20 minutes. The crude oil is from the oil
reservoir of N 2.

[0065] 5. Detecting the Bacillus in the cultures using
molecular biological techniques. A 5 ml of the culture obtained in step 4
was transferred into a sterile centrifuge tube in a sterile condition and
centrifuged at 10,000×g for 10 minutes for cell collection, removed
the supernatant and added 1 ml distilled water to resuspend the cells
sedimentation. DNA in the resuspension were extracted following the
manufacturer's protocol of the FastDNA Spin Kit for Soil, and extracted
DNA were amplified by PCR using specific 16S RNA gene primers for
bacteria to construct clone library, the representative clones was
sequenced and determined their phylogenetic affiliations.

[0066] Two 16S rRNA gene clone libraries (N2PB and N2IB) were constructed
from the two samples following the invention. 6 and 7 OTUs were
identified in N2PB and N2IB, respectively. 100% of the sequences in clone
library of N2PB and N2IB were identified as Bacillus, particularly
dominated by Bacillus halodurans and Bacillus sp. in clone library of
N2PB, and dominated by Bacillus halodurans in clone library of N2IB
(Table 9 and 10).

[0067] A phylogenic tree can be constructed by analysis of the Bacillus
sequences detected by above-identified 5 examples of the present
invention and their most relative homological sequences based on the
Basic Local Alignment Search Tool (BLAST) in the GenBank database of the
National Center for Biotechnology Information (NCBI) by DNAMMAN software
analysis. Wherein, the position of the Bacillus detected can be directly
presented in the phylogenic tree.

[0068] Most of microbes in the samples detected by the methods of the
present invention were affiliate with Bacillus sp., some of which have
low similarities with cultured strains of Bacillus, which indicated that
most of microbes detected were new species. The present invention can
simplify the step of isolation and identification of new strains of
Bacillus previously considered as uncultivable, which provides a new
method for screening potential microbes for MEOR and further provides a
research method for ecological study of the Bacillus from oil reservoirs.

[0069] While the invention has been described in conjunction with the
exemplary embodiments described above, it is to be noted that many
equivalent modifications and variations will be apparent to those skilled
in the art when given in this disclosure. Various changes to the
described embodiments may be made without departing from the spirit and
the scope of the invention.

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